Retinyl acetate retinol from

Dissolve the appropriate methyl ester in methanol, and saponify as previously described for preparation of retinol from retinyl acetate After saponification, add water, and then acidify with dilute glacial-acetic acid make sure the solution is acidic to litmus paper. (In aqueous-alkaline solution, retinoid carboxylic acids remain as sodium salts, and are not extracted by organic solvents.) Extract the retinoid-carboxylic acid with diethyl ether two or three times (Note that hexane is not a good solvent for these polar retinoids.) Then wash the ether extract with water, and dry it over anhydrous sodium sulfate. Alternatively, if the volume is small, vortex and centrifuge the sample, remove any water, and evaporate the solvent The retinoid-carboxylic acids usually are obtained as yellow solids. Do not add any (not even a trace) HCl to 5,6-epoxy retinoids, because they instantaneously undergo isomerization to 5,8-epoxy retinoids, this change in structure is readily confirmed by the change m absorption spectrum (Table 1). 

Fig. 7 Comparative HPLC separations of a standard solution of six vitamins using (A) 250 X 4.0-mm-ID standard-bore and (B) 250 X 2.0-mm-ID narrow-bore columns. Stationary phase (both columns), 5-/tm Nu-cleosil-120-5 C8 (octyl) mobile phase, methanol/water (92 8). Flow rate (A) 0.7 ml/min, (B) 0.2 ml/min. Injection volume, 1 fi 1. Wavelength-programmed absorbance detection. Peaks (1) retinol (2) retinyl acetate (3) vitamin D3 (4) a-tocopherol (5) a-tocopheryl acetate (6) retinyl palmitate. (From Ref. 108.)...

Several different quantification procedures for vitamin A have been described in the literature, some using retinol directly as a standard and some using retinyl acetate, which is converted to retinol by saponification. The latter approach is generally preferred, because crystalline all-trans-retinyl acetate is commercially available in high purity and is free from cis isomers. Commercial sources of retinol are oily preparations and are at best only about 70% pure. There are two ways of preparing a retinol standard from retinyl acetate. 

Figure 4 Reversed-phase HPLC elution profiles of tocopherols (panel A), retinoids (B), and carotenoids (C) present in human plasma (200 pL). Blood was collected 3 h after an oral dose of retinoic acid. The chromatogram was obtained by use of gradient elution (Table 3). Peak identification 2, 4-oxo-retinoic acid 4, retinoyl P-glucuronide 7, retinoic acid 8, retinol 9, retinyl acetate (internal standard) 15, butylated hydroxy toluene 16, y-tocopherol 17, a-tocopherol 18, free bilirubin 19, lutein 20, zeaxanthin 21, 2, 3 -anhydrolutein 22, P-cryptoxanthin 23, lycopene 24, a-carotene 25, P-carotene. (From Ref. 73.)...

Solvent removal and ionization are combined during electrospray in which the HPLC eluate (at flow rates as high as 1 mL/min) is sprayed through a capillary electrode at high potential to form a fine mist of charged droplets at atmospheric pressure. As the solvent evaporates, gas-phase sample ions are formed. The first application of electrospray LC-MS to the analysis of retinoids was reported by van Breemen and Huang (308). Retinoic acid, retinol, retinal, and retinyl acetate were analyzed without derivatization using a C30 reversed-phase HPLC column. Retinoic acid formed abundant deprotonated molecules, [M-H] , with a limit of detection of 23 pg injected on-column. Positive ion electrospray produced an abundant protonated molecule for retinal, and a base peak of m/z 269 was observed for retinol and retinyl acetate, which corresponded to elimination of water or acetic acid, respectively, from their protonated molecules. The limits of detection of retinal, retinol, and retinyl acetate were 1.0 ng, 0.5 ng, and 10 ng, respec-... 

Figure 4. Correlation between retinoid-induced decreases in saturation density and induction of gap junctional communication. Confluent cultures of 10 Tl/2 cells (panel A) or methylcholantlirene transformed 10 Tl/2 cells (panel B) were treated with various concentrations of retinoids for five days. At this time cells were probed by microinjection of Lucifer yellow and transfer of dye to adjacent cells was quantitated. Total cell counts were also performed at this time. Results are expressed as a percent of the untreated controls. Symbols retinol (A) retinyl acetate (A) diW-trans retinoic acid ( ) 13-cw retinoic acid ( ) TTNPB (O). The correlations were statistically highly significant, for 10 Tl/2 cells and for transformed cells, P < 0.001. Data from reference [28] with permission.

Spectroscopic methods such as uv and fluorescence have rehed on the polyene chromophore of vitamin A as a basis for analysis. Indirectly, the classical Carr-Price colorimetric test also exploits this feature and measures the amount of a transient blue complex at 620 nm which is formed when vitamin A is dehydrated in the presence of Lewis acids. For uv measurements of retinol, retinyl acetate, and retinyl palmitate, analysis is done at 325 nm. More sensitive measurements can be obtained by fluorescence. Excitation is done at 325 nm and emission at 470 nm. Although useful, all of these methods suffer from the fact that the method is not specific and any compound which has spectral characteristics similar to vitamin A will assay like the vitamin... 

Retinyl acetate [127-47-9] M 328.5, m 57". Separated from retinol by column chromatography, then crystd from MeOH. See Kofler and Rubin [Vitamins and Hormones (NY) 18 315 1960] for review of purification methods. Stored in the dark, under N2 or Ar, at 0°. See Vitamin A acetate p. 574 in Chapter 6. 

Similar processes were related. TiCl4 was added to a solution of the diol to give a crude mixture of isomers in which the 5-chlorosulfone was the main compound in 95% yield. The mixture was treated with MeOK to produce crude retinol. Acetylation with acetic anhydride (AC2O) in pyridine, in the presence of DMAP, provided the retinyl acetate in 70% from the diol [38,39], Fig. (15). 

Retinyl acetate [127-49-9] M 328.5, m 57 . Separated from retinol by column chromatography, then... 

The vitamin A value of foods has traditionally been expressed in international units (IU). One IU is defined as the amount of vitamin A activity contained in 0.334 fig of all-trans-retinyl acetate, which is equivalent to 0.300 fig of alRran.v-retinol. In 1965, an expert committee decided to abandon the IU for vitamin A, proposing instead that the vitamin A value be designated in terms of retinol equivalents (RE), expressed in micrograms of retinol. The RE is defined as the amount of retinol present plus the equivalent amount of retinol that can be obtained from the provitamins. It is purely a dietary concept and is not an equivalency in the usual chemical sense ... 

Rosenfeld et al. (144) have recorded the absorption spectrum of the fluorescent states of retinol, retinyl acetate, and retinyl-n-butylamine, using pulsed laser photolysis. Theoretical calculations (145) have closely reproudced the observed 435-nm band assuming that emission originates from the lowest - Ag state. Unfortunately, these results are not discriminative as far as identification of the lowest singlet state is concerned, since a strong absorption in the same region is also predicted for the 1B+ state (145). 

Epoxidation of methyl 7,8-dihydroretinoate (114) with monoperphthalic acid gave three main products (115)-(117). In the presence of acid (115) gave the spiro-derivative (118). The autoxidation of solid all-frans-retinyl acetate and palmitate, tra 5-axerophtene (82), and ll-cw-retinol has been studied. From amorphous samples dialkyl peroxides and carbonyl and hydroxy-compounds were formed, but in the crystalline state carbonyl compounds were the only products. ... 

FIG. 7. Observed data and model-simulated values for fraction of dose in plasma long-chain retinyl esters ( ), retinol (A), and retinyl acetate (O) vs time after intravenous administration of pH]vitamin A-labeled chylomicrons. Data are from Green et aL (1993). 

Blood collected at each time point was allowed to remain on ice for 15 min prior to centrifugation at 1800g. Plasma was transferred to 5-ml cryogenic vials and stored at -8(PC until analyzed. Plasma lipids were extracted from duplicate 2.2-g plasma aliquots for isotope ratio analysis or from 0.25-g aliquots for HPLC quantification of retinol and /3C after addition of internal standard (retinyl acetate). Plasma aliquots were de-proteinized with 1 vol of ethanol and lipids extracted with 3 vol of hexane (Optima Grade, Fisher Scientific, Rochester, NY). 

Immediately internal to this, the visceral yolk sac endoderm is rich in transcripts of cellular retinol binding protein (CRBP I) (6). The mechanism of transfer involves receptor-mediated uptake of retinol from maternal RBP-retinol by the visceral yolk sac endoderm, where it binds to CRBP I and interacts with the enzymes mediating RA synthesis (7). The assumption that retinol is retained specifically in CRBP I-expressing embryonic tissues has been verified by using 14C-labeled retinyl acetate (delivered intravenously to the pregnant dam) as a source of retinol (8). 

FIGURE 10.8 Chromatograms of a mix of standard vitamin A obtained with either HPLC (a) or UPLC (b). Detection was performed at 325 nm (12) retinol and (13) retinyl acetate. AU absorbance units. (Adapted from Chauveau-Duriot, B. et al. 2010. Anal. Bioanal. Chem. 397 777-790. With permission.)... 

The original work on which subsequent study and synthesis of retinoids are based was carried out by Karrer and Morf (1933) and Heilbron et al, (1932, 1948). In 1931 Karrer et aL were able to determine the structure of retinol (1) using a highly purified vitamin A extract that they had obtained from shark liver oil (von Euler and Karrer, 1931). Using such retinol preparations, the first oily retinol esters [for example retinyl acetate (9)] were prepared (Karrer et al., 1931 Heilbron et al., 1932). 

In 1935 Hamano and Kawakami (1935) characterized retinol (1) as the P-naphthoate and anthraquinone P-carboxylate. Later Baxter and Robeson (1940) were able for the first time to obtain crystalline retinyl palmitate (113) and crystalline retinol (1) from liver oils. Crystalline retinyl acetate (9) and crystalline retinyl succinate were obtained at a later date (Baxter and Robeson, 1942). These very pure compounds made possible the accurate determination of a number of physical data. In 1946 Hanze et al. (1948) synthesized pure retinyl methyl ether (571) from crystalline retinol (1), and the total synthesis of this ether was reported at the same time by Milas et al. (1948). At this time also, syntheses of retinoids were carried out by Isler and associates and led to the first industrial synthesis of retinol derivatives (Isler et al., 1947 Isler, 1950 Heilbron and Weedon, 1958 Isler, 1979). 

Retinol (1) was very readily obtained from commercial retinyl acetate (9) by alkali-catalyzed hydrolysis (Isler et al., 1947, 1949 Samecki et al., 1962). Reduction of esters of retinoic acid (3) with lithium aluminum hydride (Matsui et al., 1962b) and with hydrogen and Raney nickel (Organon, 1950) also gave retinol (1), this also being the product obtained when retinaldehyde (2) was reduced by various methods, for example, with sodium borohydride or lithium borohydride (Kaegi et al., 1982), aluminum isopropylate (Shchavlinskii et al., 1979), lithium aluminum hydride (Robeson et al., 1955a Pommer, 1960), or catalytic reduction over platinum(IV) oxide/cobalt(II) acetate tetrahydrate (Steiner, 1974). 

For the synthesis of retinaldehyde (2), a large number of oxidation processes have been worked out that permit (2) to be prepared in a very simple manner from commercially available retinyl acetate (9). Thus, when manganese(lV) oxide precipitated in alkaline medium was used and the reaction was carried out in petroleum ether, retinol (1) was converted to retinaldehyde (2), without unde-... 

Retinol-11,12- H2 of low specific activity has been prepared (Isler et al., 1960) based on the work of Isler et al. (1947) on the synthesis of retinyl acetate. The same sequence of reactions as shown in Fig. 8, with several improvements in technique because of the small scale, was applied by Perry et al. (1982) to the preparation of retinoic acid tritiated at very high specific activity. Pure, recrystallized diol (XXVI) was partially hydrogenated with tritium over Lindlar catalyst in the presence of quinoline and the dihydro compound (XXVII), acety-lated in such a way as to afford mainly the monoacetate (XXVIII). Exposure of the acetate at low temperature for a very short time to very dilute hydrogen bromide in methylene chloride gave, after chromatography, pure retinyl-11,12- H2 acetate (XXIXa). Simultaneous hydrolysis and oxidation of the retinyl acetate by silver oxide in aqueous methanolic sodium hydroxide then yielded all-fran -reti-noic-11,12- H2 acid (Vllh). The specific activities obtained ranged from 25 to 40 Ci/mmol. 

Because of the inherent instability of labeled retinol, the usual practice is to store this compound as a more stable ester (e.g., retinyl acetate) or to keep it in the retinoic acid stage from which it can be prepared by reduction. From esters like the acetate, retinol can be obtained by hydrolysis with base or even by reduction with lithium aluminum hydride at low temperature. For this conversion, a trans-esterification in methanol is preferred, with catalysis by trace amounts of sodium methoxide followed by purification of the retinol by HPLC (Kaegi and Bupp, 1982). 

When measuring the level of nonpolar retinoids in blood, a modified method of Thompson et al, (1971) could be used (see also Varma and Beaton, 1972 Bieri et al., 1979 Ross, 1981). Briefly, the plasma or serum (100-200 xl) is mixed with 1 volume of internal standard solution (e.g., retinyl acetate) in ethanol. To the mixture is added 1-4 volumes of hexane the contents are mixed well and then centrifuged. The hexane (upper phase) is carefully removed and used for chromatography or other analyses. For added protection against oxidation, butylated hydroxytoluene (100 xg/ml) can be added to the hexane. Other solvents used for extraction of retinol and its esters from serum include chlo-roform methanol (see, for example, DeRuyter and De Leenheer, 1978) and hexane methylene chlorideiisopropanol (Besner /., 1980). Recoveries, when reported, range near 100%. 

Fig. 4. Chromatogram of a mixture of retinyl ester standards. Column, Supelcosil LC-8 (5 jLm) mobile phase, acetonitrile water (88 12) from origin to arrow and acetonitrile water (98 2) for rest of chromatography flow rate, 3 ml/min. The esters of retinol are 1, acetate, 2 0 2, laurate, 12 0 3, 7-linolenate, 18 3 4, myristate, 14 0, 5, palmitoleate, 16 1 6, linoleate, 18 2 7, palmitate, 16 0 8, oleate, 18 1 9, stearate, 18 0 10, arachidonate, 20 4. (Reprinted with permission from Ross, 1981.)...

One of the most important phenomena for analysis of the mechanism of action of retinoids in prevention of cancer is their ability to suppress the transformation of nonneoplastic C3H/10T1/2 mouse fibroblasts that have been exposed to the chemical carcinogen 3-methylcholanthrene. This was first reported by Merriman and Bertram (1979) using retinyl acetate, retinol, and retinaldehyde as the retinoids. Similar findings were reported by Harisiadis et al. (1978), who showed that motretinid (F3) could suppress radiation-induced transformation of the same cell line. A series of retinoids has been tested by Bertram (1980 see also Bertram et al., 1982) for their ability to suppress methylcholanthrene-induced transformation effects were evaluated over a test range from approximately 10 to 10 M. Retinyl acetate, A -(4-hydroxyphenyl) retinamide (E16), and retinylidene di-medone (C6) were found to be most effective, and A -benzoylretinylamine (B2) was only slightly effective. Surprisingly, all-rran -retinoic acid and 13-cw-reti-noic acid were ineffective in these tests the reason for these results is not clear. 

Retinol or its solutions do not keep well even when kept under an inert gas at low temperature. Commercial retinol often contains substantial amounts of impurities because of its instability. Retinal and retinyl acetate are commercially readily available and are quite stable when kept under an inert gas and at low temperatures If a pure standard of retinol is not available, it can be prepared easily from either retinal or retinyl acetate as follows. 

Retmyl acetate is readily available commercially and is relatively inexpensive. Dissolve a few crystals (or a drop, if oily), 1-10 mg as appropnate, of retinyl acetate in methanol (about 1-2 mL). Add about 100 iL of NaOH solution (prepared by adding a drop of water to 1 or 2 pellets of NaOH, and then adding about 1 mL methanol shake until clear) to the retinyl-acetate solution. Make sure that the solution is alkaline to litmus paper. If necessary, add more NaOH solution (excess will not be harmful). Reflux this solution at 60°C for 15-30 min, or keep warm at 50 C for 1-2 h. Analyze the product by TLC, developing the plate as previously described, and look for the fluorescent retinol spot under a UV lamp (366 nm). When no more retinyl acetate is seen, extract the retinol from the solution by adding water (about 1 mL), and hexane... 

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